**4.3 Application of PEF for wine decontamination after malolactic fermentation**

PEF inactivation of LAB strains involved in the MLF of wine has been studied by different authors. Among the microorganisms investigated, Puértolas et al. found that *Lactobacillus plantarum* and *hilgardii* displayed the highest resistance to PEF [26]. Similarly, out of a total of 25 different wine-related microorganisms, *Oenococcus oeni* O46 and *Pediococcus pentosaceus* were found to be the ones most resistant to a PEF treatment (23 kV/cm, 95 kJ/kg, 49°C) [33]. PEF treatments of 20 kV/cm and 320 kJ/kg were capable of inactivating up to 5.0 log cycles of *O. oeni* with a temperature remaining below 15°C [40].

Few studies have been conducted on the inactivation of microorganisms after malolactic fermentation. González-Arenzana et al. observed that after the MLF of three wines, the application of a PEF treatment (95 kJ/kg, 23 kV/cm) in combination with a low SO2 concentration (15 mg/L) had similar or even greater effectivity than an increased dose of SO2 (30 mg/L) in the microbial stabilization of wine [41]. PEF treatments alone, or combined with SO2, allowed for a significant reduction in the overall population of the main microbial strains of yeasts, LAB, and ABB. Moreover, stabilization by PEF treatments was effective in inhibiting microbial growth after six months of storage, with no changes in physicochemical and sensory properties in comparison to wines stabilized by SO2.

### **4.4 Application of PEF for wine decontamination before aging in barrels**

Aging in oak barrels is one of the key steps in the production of high-quality wine, due to its gradual development in terms of aroma, color, and stability [42]. Oak wood is a porous material that is necessary for air exchange and for the maintenance of low oxidation conditions in wine during the aging process, but oak wood barrels are extremely difficult to clean and sanitize. They therefore present an ideal niche for microbial proliferation, and can be a source of contamination for subsequent batches of wine [43]. This is a great concern in wineries – especially in the case of *Brettanomyces* colonization, due to that yeast's negative impact on wine quality, along with the difficulty of early identification and the considerable economic losses associated with its proliferation. Many other microbial strains can colonize the oak barrels and become a source of contamination and wine spoilage. Any strategy for the microbial decontamination of aged wine in barrels should nevertheless preserve all the quality parameters acquired during this long and expensive process.

Aged preservative-free wine in oak barrels was successfully treated by PEF, with a high-level reduction in the population of the main naturally present strains [44]. However, the recovery of some of the main microorganisms involved in aging was observed in control and PEF-treated wines after 5–9 months of storage. Therefore, different PEF parameters should be tested in order to optimize PEF conditions in this scantly investigated step of winemaking. Further studies regarding the effect of PEF treatments on valuable aging characteristics prior to bottling should be carried out, as well as on the evolution of the microbial population during these long storage periods.
